THE CONSTRUCTION OF A REPRESENTATIVE HUMAN CRYOPRESERVED HEPATOCYTE POOL FOR METABOLISM STUDY
Zhihong Zhang O’Brien*, Troy Bremer, Kevin Holme and Yong Hee Lee*1
In Vitro System, Lion Bioscience, San Diego, CA 92121
ABSTRACT
RESULTS
Table 1. IVT 51-donor enzyme activity database
Due to the extensive inter-individual difference and polymorphic distribution of liver enzymes, it is necessary to construct
a representative human cryopreserved hepatocyte (HCH) pool for metabolism study. To obtain a representative HCH
pool, enzyme activity database of total 51 donors (In Vitro Technology (IVT)) was analyzed using statistical tools. Due to
the non-normal distribution of all isozymes from vendor database, a median value, its 99% confidence interval (CI) and
confidence intervals of 67% typical population for each isozyme were defined. To select a representative pool and
minimize the pool-to-pool variation, the following criteria will be applied: 1) at least 80% of individual isozyme activity
values must be within their corresponding acceptable activity range; 2) no more than 30-35% of each enzyme activity
value/X donors should be outside the acceptable activity range; and 3) mean activity for each enzyme /pool should fall
into 99% CI of its corresponding median value. By doing so, it is expected to result in approximate one- to three-fold
(except 4-fold for CYP1A2 and 6-fold for CYP2C19) variations of isozyme activities among pools. Thirty 6-donor pools
were constructed based on the selection criteria described above, and four of them were tested for isozyme activities
using 11 functional probe substrates. Our results showed that: 1) good correlation (0.8slope1.3 and R20.8) is
achieved between the mean of each isozyme activity per pool and median values of each enzyme in the database; 2)
enzyme activity changing trends of the virtual HCH pools constructed through IVT database are compatible with our
actual results; and 3) one- to three-fold isozyme activity differences are observed among pools.
INTRODUCTION
Hepatocyte has been recognized as one of the most powerful in vitro tools in metabolism and toxicity studies. The
advent of cryopreservation technologies opened a new field for using hepatocyte more conveniently and wisely,
especially for human hepatocyte. Cryopreservation techniqe circumvents the huge waste of freshly isolated human
hepatocytes due to the lack of appropriate storage, and provides a tremendous opportunity to minimize the interindividual differences and polymorphic distribution of liver enzymes. To maintain the consistency of liver enzyme
activities and reduce the inter-indivadual enzyme differences in hepatocyte system used in each study, it is better to
construct a uniformed hepatocyte pool. However, it is impossible to have a same generic pool for each indivadual
study. Then, what would be the reasonable way for the hepatocyte pool construction? Up to now, there hasn’t been
any strong interest shown in how to construct the best pool although almost everyone realizes the importance of doing
so. Recently, our lab started with a total of 51 human cryopreserved hepatocyte donor database published by In Vitro
Technology (IVT). To initiate the investigation, the enzyme activity database of 51 donors was analyzed by using
statistical tools. Based on the statistical results, hepatocyte pool selection criteria was established. Thirty-four 6-donor
pools were constructed according to the selection criteria and four of them were actually tested in our lab for isozyme
activities using 11 functional marker compounds. This study provides a basis for obtaining a representative human
cryopreserved hepatocyte (HCH) pool to reduce the chance of causing enzyme activity variation during each study.
Lot #
Viability
51
70
83
86
87
89
90
91
94
98
99
102
104
105
107
110
111
113
114
117
118
122
129
133
ACU
CPN
DRL
EFA
ENR
EQB
ET R
EVY
GAL
GIU
GNG
GUY
HRK
IEM
KMI
KPT
KRM
KT G
MAN
MQF
MYO
NQT
OCF
OQD
PFM
RKB
RNG
65
74
59
74
71
77
74
73
67
72
86
81
81
67
81
77
71
60
75
85
65
70
90
82
81
81
84
69
73
88
88
80
64
84
70
92
63
88
74
83
78
94
97
91
94
85
82
94
87
63
91
COUM
2A6
55
36
29
51
34
25
30
13
41
75
21
42
63
59
60
45
4
15
39
42
81
26
4
67
53
28
25
9
69
14
30
2
52
87
57
65
69
34
32
9
126
135
31
79
40
76
91
80
21
42
119
Conventional pool construction for liver fractions
•
•
Human microsome and S9: 10 to 15 donors as an optimum pool
Human cryopreserved hepatocyte: Not addressed yet
Advantages for construction of a representative human cryopreserved hepatocyte pool
•
•
•
Reduce the interindividual variations in human liver enzymes
Make experimental results more comparible between studies or labs
Provide experimental data more representation of a “generic human”
Study Design:
(1) Statistical analysis of IVT 51-donor database to establish the pool selection criteria:
•
Determine type of distribution of 51-donors for each isozyme
•
Define median value and its 99% confidence interval (CI) for each isozyme
•
Define confidence intervals of 67% typical population for each isozyme
(2) Construction of thirty-four HCH pools based on the pool selection criteria
(3) Actual pool comparisons by measuring isozyme activities using 11 functional markers
ECOD
2E1
17
22
42
10
43
8
7
66
37
24
7
27
44
24
24
35
9
34
21
21
56
29
27
24
25
40
27
35
27
18
34
23
27
29
33
11
55
23
29
32
55
86
35
29
9
39
26
39
11
16
97
7-HCG
GT
47
88
190
50
81
23
13
44
24
57
4
8
247
38
21
23
2
16
10
60
208
80
67
129
74
100
100
47
124
65
13
218
59
55
54
73
124
129
192
248
83
111
119
10
24
80
98
233
67
100
298
7-HCS
ST
7
33
25
9
35
6
0
10
4
12
0
13
25
14
0
4
3
0
5
25
29
5
18
19
18
13
10
5
31
9
13
77
6
13
8
12
12
34
18
30
17
25
32
12
0
23
17
26
10
8
27
MEPH
2C19
1
12
21
1
3
1
2
12
21
9
1
1
2
1
16
6
3
4
5
16
26
1
1
27
8
2
4
18
107
31
7
33
3
171
5
20
23
72
6
55
103
12
3
2
0
2
52
10
3
8
177
T EST
3A4
115
55
74
38
56
11
19
252
126
45
60
7
58
29
234
206
147
151
59
113
208
42
16
96
80
168
43
66
77
49
13
24
94
86
95
90
136
48
69
65
98
60
40
85
12
151
147
135
116
36
207
T OLB
2C9
10
32
39
13
50
13
15
36
40
25
10
6
37
27
41
11
2
51
T BD
45
51
23
33
11
16
14
31
12
36
14
13
25
13
28
22
13
34
23
9
26
46
32
14
5
7
14
33
11
8
17
34
PHEN
1A2
6
43
33
0
4
0
0
27
19
12
0
6
8
12
7
54
19
25
3
8
39
4
10
24
4
37
37
47
38
21
37
17
57
20
16
5
64
19
14
56
74
74
6
7
0
20
40
14
15
20
71
CZX
2E1
23
40
30
18
64
29
16
36
87
32
22
37
20
27
48
36
19
74
45
25
13
25
51
32
23
21
31
67
32
35
69
38
23
33
22
8
22
34
18
32
44
66
34
46
11
34
32
28
33
19
41
Gender
F
M
M
F
F
F
F
F
F
F
F
M
M
M
F
F
F
F
F
F
F
M
F
F
F
M
M
M
M
M
F
M
F
M
F
M
F
M
M
F
F
M
M
F
F
M
F
M
F
F
F
Phenacetine (PHEN)
Coumarin (COUM)
Tolbutamide (TOLB)
(S)-Mephenytoin (MEPH)
Dextromethorphan (DEX)
Chlorzoxazone (CZX)
Midazolam (MID)
7-Hydroxycoumarin (7-HC)
Substrate Conc. #1
(M)a
250
100
500
250
50
250
10
250
Methylumbelliferone (MUF)
500
Trans-4-phenyl-3-butene-2one (TPBO)
500
7-Ethoxycoumarin (7-EC)
75
a
(1) Statistical analysis of IVT 51-donor database to establish the pool selection criteria:
Table 3. Median confidence range and acceptable activity range of indivadual isozyme
Substrate:
COUM
DEX
ECOD
7-HCG
7-HCS
MEPH
TEST
TOLB
PHEN
CZX
Enzyme:
2A6
2D6
2E1
GT
ST
2C19
3A4
2C9
1A2
2E1
Median
confidence
range:
30-60
12-27
25-41
50-117
8-20
3-18
49-113
13-34
8-33
23-36
Acceptable
activity range:
21-76
9-33
11-43
8-184
5-27
1-31
25-147
10-46
4-43
20-47
Donor selection criteria:
• At least 80% of individual isozyme activity values must be within their corresponding acceptable range.
• No more than 30-35% of each enzyme activity value/X donors should be outside the acceptable activity
range.
• Mean activity for each isozyme/pool should fall into its corresponding median confidence range.
(2) Construction of thirty-four HCH pools based on the pool selection criteria:
Good correlation (0.8slope1.3 and R20.8) is achieved between the mean of each isozyme activity per pool and
median values of each enzyme in the database.
One- to three-fold isozyme activity differences are observed among pools.
•
•
(3) Actual pool comparisons by measuring isozyme activities using 11 functional markers:
Enzyme activity values are based
on IVT database
Table 2. Eleven functional marker compounds
Substrate ID
METHODS
DEX
2D6
9
4
24
23
36
21
25
29
12
5
15
40
21
15
28
24
10
47
23
16
34
30
24
16
8
6
7
9
28
11
1
20
7
15
17
12
11
17
11
12
36
40
12
17
24
12
16
13
1
16
14
Metabolites Monitored
Detection
Acetaminophen
7-Hydroxycoumarin
OH-Tolbutamide
OH-mehpenytoin
Dextrorphan
OH-Chlorzoxazone
1’- and 4’-OH Midazolam
7-Hydroxycoumarin
glucuronide & 7hydroxycoumarin sulfate
Methylumbelliferone
glucuronide &
methylumbelliferone
sulfate
Trans-4-phenyl-3-butene2-one glutathione
conjugate
7-Hydroxycoumarin
7-Hydroxycoumarin
glucuronide
7-Hydroxycoumarin
sulfate
LC/MS/MS
LC/UV
LC/MS/MS
LC/MS/MS
LC/MS/MS
LC/MS/MS
LC/MS/MS
LC/UV
Enzyme activity values are based
on Lion actual experimental data
LC/UV
LC/MS/MS
•
LC/UV
This saturated concentration (>> Km) was used for the measurement of metabolite formation rates.
Enzyme activity changing trends of the virtual HCH pools constructed through IVT database are compatible with
our actal results.
CONCLUSION
•
The donor selection criteria based on the statistical analysis successfully provides a basis for obtaining a
representative human cryopreserved hepatocyte (HCH) pool.